Alpha-hemolysin variants and uses thereof

ABSTRACT

Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. Provisional PatentApplication Ser. No. 62/325,749, filed Apr. 21, 2016, and titled“Alpha-Hemolysin Variants and Uses Thereof.” The entire disclosure ofthe above-identified priority application is hereby fully incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 17, 2017, isnamed P33551-US1_SL.txt and is 42,861 bytes in size.

TECHNICAL FIELD

Disclosed are compositions and methods relating to Staphylococcalaureaus alpha-hemolysin variants. The alpha-hemolysin (α-HL) variantsare useful, for example, as a nanopore component in a device fordetermining polymer sequence information. The nanopores, methods, andsystems described herein provide quantitative detection of single strandnucleic acids, such as DNA, RNA, etc., employing nanopore-basedsingle-molecule technology with improved characteristics.

BACKGROUND

Hemolysins are members of a family of protein toxins that are producedby a wide variety of organisms. Some hemolysins, for example alphahemolysins, can disrupt the integrity of a cell membrane (e.g., a hostcell membrane) by forming a pore or channel in the membrane. Pores orchannels that are formed in a membrane by pore forming proteins can beused to transport certain polymers (e.g., polypeptides orpolynucleotides) from one side of a membrane to the other.

Alpha-hemolysin (α-HL, a-HL or alpha-HL) is a self-assembling toxinwhich forms a channel in the membrane of a host cell. Alpha-HL hasbecome a principal component for the nanopore sequencing community. Ithas many advantageous properties including high stability,self-assembly, and a pore diameter which is wide enough to accommodatesingle stranded DNA but not double stranded DNA (Kasianowicz et al.,1996).

Previous work on DNA detection in the a-HL pore has focused on analyzingthe ionic current signature as DNA translocates through the pore(Kasianowicz et al., 1996, Akeson et al., 1999, Meller et al., 2001), avery difficult task given the translocation rate (˜1 nt/μs at 100 mV)and the inherent noise in the ionic current signal. Higher specificityhas been achieved in nanopore-based sensors by incorporation of probemolecules permanently tethered to the interior of the pore (Howorka etal., 2001a and Howorka et al., 2001b; Movileanu et al., 2000).

The wild-type a-HL results in significant number of deletion errors,i.e. bases are not measured. Therefore, α-HL nanopores with improvedproperties are desired.

BRIEF SUMMARY OF THE INVENTION

As described herein, provided are mutant staphylococcal alpha hemolysin(αHL) polypeptide containing an amino acid variation that enhances thetime to thread, e.g., decreases the time to capture of the molecule ofinterest. For example, the disclosed variants reduce the time to threadof the molecule of interest, e.g., various tagged nucleotides or anucleotide to be sequenced.

In certain example aspects, the α-hemolysin (α-HL) variants comprise asubstitution at a position corresponding to any one of V149K, E287R,H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof of SEQID NO: 14 (the mature, wild-type alpha hemolysin sequence). Thesubstitution of the α-hemolysin may also be a positive charge. Theα-hemolysin variant may also include a substitution at H144A of SEQ IDNO: 14. The α-hemolysin variant may also, in certain aspects, includeone or more one or more glycine residues at residues 127-131 of SEQ IDNO: 14, such as a series of glycine residues that span the entire lengthof residues 127 through 131 of SEQ ID NO: 14.

In certain example aspects, the α-hemolysin variant includes an aminoacid sequence having at least one of the substitutions described herein,while the sequence of the α-hemolysin variant has at least 80%, 90%,95%, 98%, or more sequence identity to the amino acid sequence set forthas SEQ ID NO: 14. In certain example aspects, the α-hemolysin variantincludes an amino acid sequence having at least 80%, 90%, 95%, 98%, ormore sequence identity to the amino acid sequence set forth as SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

In certain example aspects, the alpha-hemolysin variant may include asubstitution corresponding to H35G+V149K of SEQ ID NO: 14. Additionallyor alternatively, the alpha-hemolysin variant may include a substitutioncorresponding to V149K+E287R+H35G of SEQ ID NO: 14. Additionally oralternatively, the alpha-hemolysin variant may include a substitutioncorresponding to V149K+E287R of SEQ ID NO: 14. Additionally oralternatively, the alpha-hemolysin variant may include a substitutioncorresponding to T109K+H35G of SEQ ID NO: 14. Additionally oralternatively, the alpha-hemolysin variant may include a substitutioncorresponding to P151K+H35G of SEQ ID NO: 14. Additionally oralternatively, the alpha-hemolysin variant may include a substitutioncorresponding to V149K+P151K+H35G of SEQ ID NO: 14. Additionally oralternatively, the alpha-hemolysin variant may include a substitutioncorresponding to T109K+V149K+H35G of SEQ ID NO: 14. Additionally oralternatively, the alpha-hemolysin variant may include a substitution ata position corresponding to V149K+K147N+E111N+127-131G+M113A+H35G of SEQID NO: 14. Additionally or alternatively, the alpha-hemolysin variantmay include a substitution corresponding toV149K+K147N+E111N+127-131G+M113A of SEQ ID NO: 14. Additionally oralternatively, the alpha-hemolysin variant may include a substitutioncorresponding to T109K+V149K+P151K+H35G. In certain example aspects, anysuch combinations may also include a substitution at H144A of SEQ ID NO:14.

In certain example aspects, the amino acid substitution described hereinallows the addition of heterologous molecules, such as polyethyleneglycol (PEG). In certain example aspects, the a-HL variant has one ormore post-translational modifications. In certain example aspects, thesubstitution is a non-native amino acid that is basic or positivelycharged at a pH from about 5 to about 8.5.

In certain example aspects, the alpha-hemolysin variant described hereinis bound to a DNA polymerase, such as via a covalent bond. For example,the alpha-hemolysin variant is bound to the DNA polymerase via aSpyTag/SpyCatcher linkage. In certain example aspects, thealpha-hemolysin variant is bound to the DNA polymerase via an isopeptidebond.

In certain example aspects, provided is a heptameric nanopore assembly.The assembly, for example, includes at least one or more of thealpha-hemolysin variants described herein. For example, the heptamericnanopore assembly may include one or more alpha-hemolysin moleculeshaving a substitution at V149K, E287R, H35G, T109K, P151K, K147N, E111N,M113A, or combinations thereof of SEQ ID NO: 14, such as describedherein.

In certain example aspects, provided is a heteromeric pore assemblyincluding a mutant αHL polypeptide (M), e.g., a pore assembly whichcontains a wild type (WT) staphylococcal αHL polypeptide and a mutantαHL polypeptide in which an amino acid variant (as provided for herein)of the mutant αHL polypeptide occupies a position in a transmembranechannel of the pore structure. For example, the ratio of WT and variantαHL polypeptides is expressed by the formula WT_(7-n)M_(n), where n is1, 2, 3, 4, 5, 6, or 7. In certain aspects, the ratio of αHLpolypeptides in the heteroheptamer is WT_(7-n)M_(n). In other aspects,the ratio is WT₆M₁. Homomeric pores in which each subunit of theheptomer is a mutated αHL polypeptide (i.e., where n=7) are alsoencompassed by the disclosure provided herein.

The nanopore protein assemblies described herein, for example, can havean altered time to thread (TTT) relative to a pore complex consisting ofnative (wild type) alpha-hemolysin. For example, the TTT may bedecreased, such as by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% ormore as compared to a heptameric nanopore assembly including native(wild type) alpha-hemolysins.

In certain example aspects, also provided are nucleic acids encoding anyof the alpha hemolysin variants described herein. For example, thenucleic acid sequence can be derived from Staphylococcus aureus (SEQ IDNO: 1). Also provided, in certain example aspects, are vectors thatinclude an any such nucleic acids encoding any one of the hemolysinvariants described herein. Also provided is a host cell that istransformed with the vector.

In certain example aspects, provided is a method of producing analpha-hemolysin variant as descried herein. The method includes, forexample, the steps of culturing a host cell including the vector in asuitable culture medium under suitable conditions to producealpha-hemolysin variant. The variant is then obtained from the cultureusing methods known in the art.

In certain example aspects, provided is a method of detecting a targetmolecule. The method includes, for example, providing a chip comprisinga nanopore assembly as described herein in a membrane that is disposedadjacent or in proximity to a sensing electrode. The method thenincludes directing a nucleic acid molecule through the nanopore. Thenucleic acid molecule is associated with a reporter molecule andincludes an address region and a probe region. The reporter molecule isassociated with the nucleic acid molecule at the probe region and iscoupled to a target molecule. The method further involves sequencing theaddress region while said nucleic acid molecule is directed through saidnanopore to determine a nucleic acid sequence of said address region.The target molecule is identified, with the aid of a computer processor,based upon a nucleic acid sequence of the determined address regiondetermined.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the time-to-thread for control heptamericnanopores as compared to heptameric nanopores including onealpha-hemolysin/phi29 DNA Polymerase conjugate and six alpha-hemolysinvariants, each variant having substitutions at H35G+V149K+H144A (i.e., a1:6 ratio), as set forth in SEQ ID NO: 4. The control nanopores (labeled“WT”) include a 1:6 ratio of alpha-hemolysin/phi29 DNA Polymeraseconjugate to six wild-type alpha-hemolysins. These data are combinedfrom many pores which were capturing the tagged nucleotides indicatingthe pore had both a polymerase and a template DNA molecule. As shown foreach tag (corresponding to A, C, G, T), the threading rate for ananopore including the variant alpha hemolysin was significantlyincreased compared to the control nanopore, thus evidencing an improved(decreased) time-to-thread. In the case of the C-nucleotide tag thethreading rate increased from a mean value of 221.62 s⁻¹ (standarddeviation 13.6 s⁻¹) to 663.15 (standard deviation 172 s⁻¹). Othernucleotide tags showed similar increases as shown in FIG. 1.

FIG. 2 is a graph showing the raw data used to generate FIG. 1. FIG. 2represents an analog to digital converter (ADC) value for our AC coupledsystem, which gives rise to an ADC value for open channel when theapplied bias is positive (175 ADC units) and when the applied bias isnegative (45 ADC units). When a tagged nucleotide is threaded into thepore, the ADC value during the application of positive bias decreasesfrom the open channel level to 145 ADC units. Several examples of thisare shown from 1020 to 1040 s in the expanded lower panel of FIG. 2.When the applied bias is negative, the tagged nucleotide exits the pore,which is why the negative open channel level is not significantlyreduced. In order to calculate the threading rate, a distribution of thetimes required in each positive bias period for the ADC value to reach athreaded level is counted for cycles whose immediately prior cycle endedat a threaded ADC value. This histogram is then fit to a standard singleexponential function, whose decay rate is the threading rate.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Practitioners areparticularly directed to Sambrook et al., 1989, and Ausubel F M et al.,1993, for definitions and terms of the art. It is to be understood thatthis invention is not limited to the particular methodology, protocols,and reagents described, as these may vary.

Numeric ranges are inclusive of the numbers defining the range. The termabout is used herein to mean plus or minus ten percent (10%) of a value.For example, “about 100” refers to any number between 90 and 110.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

Definitions

Alpha-hemolysin: As used herein, “alpha-hemolysin,” “α-hemolysin,”“a-HL” and “α-HL” are used interchangeably and refer to the monomericprotein that self-assembles into a heptameric water-filled transmembranechannel (i.e., nanopore). Depending on context, the term may also referto the transmembrane channel formed by seven monomeric proteins.

Amino acid: As used herein, the term “amino acid,” in its broadestsense, refers to any compound and/or substance that can be incorporatedinto a polypeptide chain. In some embodiments, an amino acid has thegeneral structure H₂N—C(H)(R)—COOH. In some embodiments, an amino acidis a naturally-occurring amino acid. In some embodiments, an amino acidis a synthetic amino acid; in some embodiments, an amino acid is aD-amino acid; in some embodiments, an amino acid is an L-amino acid.“Standard amino acid” refers to any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acid”refers to any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or obtained from anatural source. As used herein, “synthetic amino acid” or “non-naturalamino acid” encompasses chemically modified amino acids, including butnot limited to salts, amino acid derivatives (such as amides), and/orsubstitutions. Amino acids, including carboxy- and/or amino-terminalamino acids in peptides, can be modified by methylation, amidation,acetylation, and/or substitution with other chemical without adverselyaffecting their activity. Amino acids may participate in a disulfidebond. The term “amino acid” is used interchangeably with “amino acidresidue,” and may refer to a free amino acid and/or to an amino acidresidue of a peptide. It will be apparent from the context in which theterm is used whether it refers to a free amino acid or a residue of apeptide. It should be noted that all amino acid residue sequences arerepresented herein by formulae whose left and right orientation is inthe conventional direction of amino-terminus to carboxy-terminus.

Base Pair (bp): As used herein, base pair refers to a partnership ofadenine (A) with thymine (T), adenine (A) with uracil (U) or of cytosine(C) with guanine (G) in a double stranded nucleic acid.

Complementary: As used herein, the term “complementary” refers to thebroad concept of sequence complementarity between regions of twopolynucleotide strands or between two nucleotides through base-pairing.It is known that an adenine nucleotide is capable of forming specifichydrogen bonds (“base pairing”) with a nucleotide which is thymine oruracil. Similarly, it is known that a cytosine nucleotide is capable ofbase pairing with a guanine nucleotide.

Expression cassette: An “expression cassette” or “expression vector” isa nucleic acid construct generated recombinantly or synthetically, witha series of specified nucleic acid elements that permit transcription ofa particular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

Heterologous: A “heterologous” nucleic acid construct or sequence has aportion of the sequence which is not native to the cell in which it isexpressed. Heterologous, with respect to a control sequence refers to acontrol sequence (i.e. promoter or enhancer) that does not function innature to regulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,transformation, microinjection, electroporation, or the like. A“heterologous” nucleic acid construct may contain a control sequence/DNAcoding sequence combination that is the same as, or different from acontrol sequence/DNA coding sequence combination found in the nativecell.

Host cell: By the term “host cell” is meant a cell that contains avector and supports the replication, and/or transcription ortranscription and translation (expression) of the expression construct.Host cells for use in the present invention can be prokaryotic cells,such as E. coli or Bacillus subtilus, or eukaryotic cells such as yeast,plant, insect, amphibian, or mammalian cells. In general, host cells areprokaryotic, e.g., E. coli.

Isolated: An “isolated” molecule is a nucleic acid molecule that isseparated from at least one other molecule with which it is ordinarilyassociated, for example, in its natural environment. An isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

Modified alpha-hemolysin: As used herein, the term “modifiedalpha-hemolysin” refers to an alpha-hemolysin originated from another(i.e., parental) alpha-hemolysin and contains one or more amino acidalterations (e.g., amino acid substitution, deletion, or insertion)compared to the parental alpha-hemolysin. In some embodiments, amodified alpha-hemolysin of the invention is originated or modified froma naturally-occurring or wild-type alpha-hemolysin. In some embodiments,a modified alpha-hemolysin of the invention is originated or modifiedfrom a recombinant or engineered alpha-hemolysin including, but notlimited to, chimeric alpha-hemolysin, fusion alpha-hemolysin or anothermodified alpha-hemolysin. Typically, a modified alpha-hemolysin has atleast one changed phenotype compared to the parental alpha-hemolysin.

Mutation: As used herein, the term “mutation” refers to a changeintroduced into a parental sequence, including, but not limited to,substitutions, insertions, deletions (including truncations). Theconsequences of a mutation include, but are not limited to, the creationof a new character, property, function, phenotype or trait not found inthe protein encoded by the parental sequence.

Nanopore: The term “nanopore,” as used herein, generally refers to apore, channel or passage formed or otherwise provided in a membrane. Amembrane may be an organic membrane, such as a lipid bilayer, or asynthetic membrane, such as a membrane formed of a polymeric material.The membrane may be a polymeric material. The nanopore may be disposedadjacent or in proximity to a sensing circuit or an electrode coupled toa sensing circuit, such as, for example, a complementary metal-oxidesemiconductor (CMOS) or field effect transistor (FET) circuit. In someexamples, a nanopore has a characteristic width or diameter on the orderof 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins.Alpha-hemolysin is an example of a protein nanopore.

Nucleic Acid Molecule: The term “nucleic acid molecule” includes RNA,DNA and cDNA molecules. It will be understood that, as a result of thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding a given protein such as alpha-hemolysin and/or variants thereofmay be produced. The present invention contemplates every possiblevariant nucleotide sequence, encoding variant alpha-hemolysin, all ofwhich are possible given the degeneracy of the genetic code.

Promoter: As used herein, the term “promoter” refers to a nucleic acidsequence that functions to direct transcription of a downstream gene.The promoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given gene.In general, the transcriptional and translational regulatory sequencesinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences.

Purified: As used herein, “purified” means that a molecule is present ina sample at a concentration of at least 95% by weight, or at least 98%by weight of the sample in which it is contained.

Purifying: As used herein, the term “purifying” generally refers tosubjecting transgenic nucleic acid or protein containing cells tobiochemical purification and/or column chromatography.

Tag: As used herein, the term “tag” refers to a detectable moiety thatmay be atoms or molecules, or a collection of atoms or molecules. A tagmay provide an optical, electrochemical, magnetic, or electrostatic(e.g., inductive, capacitive) signature, which signature may be detectedwith the aid of a nanopore. Typically, when a nucleotide is attached tothe tag it is called a “Tagged Nucleotide,” The tag may be attached tothe nucleotide via the phosphate moiety.

Time-To-Thread: The term “time to thread” or “TTT” means the time ittakes the polymerase-tag complex or a nucleic acid strand to thread thetag into the barrel of the nanopore.

Variant: As used herein, the term “variant” refers to a modified proteinwhich displays altered characteristics when compared to the parentalprotein, e.g., altered ionic conductance.

Variant hemolysin: The term “variant hemolysin gene” or “varianthemolysin” means, respectively, that the nucleic acid sequence of thealpha-hemolysin gene from Staphylococcus aureus has been altered byremoving, adding, and/or manipulating the coding sequence or the aminoacid sequence of the expressed protein has been modified consistent withthe invention described herein.

Vector: As used herein, the term “vector” refers to a nucleic acidconstruct designed for transfer between different host cells. An“expression vector” refers to a vector that has the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are commerciallyavailable. Selection of appropriate expression vectors is within theknowledge of those having skill in the art.

Wild-type: As used herein, the term “wild-type” refers to a gene or geneproduct which has the characteristics of that gene or gene product whenisolated from a naturally-occurring source.

Percent homology: The term “% homology” is used interchangeably hereinwith the term “% identity” herein and refers to the level of nucleicacid or amino acid sequence identity between the nucleic acid sequencethat encodes any one of the inventive polypeptides or the inventivepolypeptide's amino acid sequence, when aligned using a sequencealignment program. For example, as used herein, 80% homology means thesame thing as 80% sequence identity determined by a defined algorithm,and accordingly a homologue of a given sequence has greater than 80%sequence identity over a length of the given sequence. Exemplary levelsof sequence identity include, but are not limited to, 80, 85, 90, 95,98% or more sequence identity to a given sequence, e.g., the codingsequence for any one of the inventive polypeptides, as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet. See also, Altschul, et al., 1990 andAltschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402, 1997.)

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 13.0.7, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used.

For ease of reference, variants of the application are described by useof the following nomenclature: Original amino acid(s); position(s);substituted amino acid(s). According to this nomenclature, for instance,the substitution of a valine by a lysine in position 149 is shown as:

-   -   Val149Lys or V149K    -   Multiple mutations are separated by plus signs, such as:    -   His35Gly+Val149Lys or H35G+V149K        representing mutations in positions 35 and 149 substituting        glycine for histidine and lysine for valine, respectively. Spans        of amino acid substitutions are represented by a dash, such as a        span of glycine residues from residue 127 to 131 being:        127-131Gly or 127-133G.        Site-Directed Mutagenesis of Alpha-Hemolysin

Staphylococcus aureus alpha hemolysin wild type sequences are providedherein (SEQ ID NO:1, nucleic acid coding region; SEQ ID NO:14, proteincoding region) and available elsewhere (National Center forBioinformatics or GenBank Accession Numbers M90536 and AAA26598).

Point mutations may be introduced by any method known in the art. Forexample, a point mutation may be made using QuikChange Lightning 2 kit(Stategene/Agilent) following manufacturer's instructions.

Primers can be ordered from commercial companies, e.g., IDT DNA.

Alpha-Hemolysin Variants

The alpha-hemolysin variants provided herein include specificsubstitutions, or one or more combination of substitutions, that improvethe time-to-thread in a nanopore-based, sequencing reaction. Byimproving the time-to-thread, high accuracy DNA sequencing can beachieved with fewer deletions in the determined sequence.

In certain example embodiments, the alpha-hemolysin variant providedherein includes one or more mutations at one or more of the locations ofthe amino acid sequence set forth as SEQ ID NO: 14. For example, any oneof the residues identified in Table 1, or combinations thereof, may bemutated to form an alpha-hemolysin variant. In certain exampleembodiments, the alpha-hemolysin variant formed from muting one or moreof the amino acids of SEQ ID NO:14 identified in Table 1 has 80%, 85%,90%, 95%, 98% or more sequence identity to the sequence set forth as SEQID NO: 14. In certain example embodiments, the mutation results in theaddition of a positive charge. For example, the mutation may result in asubstitution of an amino acid residue identified in Table 1 to anarginine, lysine, histidine, asparagine, or other amino acid that cancarry a positive charge.

In certain example embodiments, the mutation includes a particularsubstitution. For example, the variant may include an amino acidsubstitution of any one of V149K, E287R, H35G, T109K, P151K, K147N,E111N, M113A, or combinations thereof of SEQ ID NO: 14. In other exampleembodiments, the variant may include one or more these samesubstitutions, while the overall sequence can have up to 80%, 85%, 90%,95%, 98% or more sequence identity to the amino acid sequence set forthas SEQ ID NO: 14. In certain example embodiments, one or more of thefirst 17 amino acids of SEQ ID NO: 14 mutated to either an A, N, K, orcombinations thereof.

To improve nanopore stability, for example, any of the alpha-hemolysinvariants described herein may also include an amino acid substitution atH144A of SEQ ID NO: 14. Additionally or alternatively, any of thevariants may include a series of glycine residue substitutions spanningfrom residue 127 to residue 131 of the sequence set forth as SEQ ID NO:14.

TABLE 1Residues of mature alpha-hemolysin that can be mutated to form alpha-hemolysin variant.Position* Residue Position Residue Position Residue Position ResiduePosition Residue  1 ALA  65 TYR 124 VAL 175 VAL 229 ALA  2 ASP  66 ARG125 THR 176 ASN 235 ASP  3 SER  67 VAL 126 GLY 177 GLN 236 ARG  4 ASP 68 TYR 127 ASP 178 ASN 237 LYS  5 ILE  69 SER 128 ASP 179 TRP 238 ALA 6 ASN  70 GLU 129 THR 180 GLY 239 SER  8 LYS  71 GLU 130 GLY 181 PRO240 LYS  9 THR  72 GLY 131 LYS 182 TYR 241 GLN 10 GLY  73 ALA 132 ILE183 ASP 244 ASN 11 THR  74 ASN 134 GLY 184 ARG 246 ASP 13 ASP  75 LYS135 LEU 185 ASP 250 GLU 14 ILE  79 ALA 136 ILE 186 SER 252 VAL 15 GLY 82 SER 137 GLY 187 TRP 253 ARG 16 SER  83 ALA 138 ALA 188 ASN 255 ASP17 ASN  85 LYS 139 ASN 189 PRO 257 GLN 18 THR  87 GLN 140 VAL 190 VAL259 HIS 19 THR  89 GLN 141 SER 191 TYR 260 TRP 20 VAL  90 LEU 142 ILE193 ASN 261 THR 21 LYS  91 PRO 143 GLY 194 GLN 262 SER 22 THR  92 ASP144 HIS 197 MET 263 THR 24 ASP  93 ASN 145 THR 198 LYS 264 ASN 25 LEU 94 GLU 146 LEU 199 THR 266 LYS 26 VAL  95 VAL 147 LYS 200 ARG 268 THR27 THR  97 GLN 148 TYR 201 ASN 269 ASN 28 TYR 102 TYR 149 VAL 202 GLY270 THR 29 ASP 103 PRO 150 GLN 203 SER 271 LYS 30 LYS 104 ARG 151 PRO204 MET 272 ASP 31 GLU 105 ASN 152 ASP 205 LYS 273 LYS 32 ASN 106 SER153 PHE 207 ALA 274 TRP 33 GLY 107 ILE 154 LYS 208 ASP 275 THR 35 HIS108 ASP 155 THR 210 PHE 276 ASP 36 LYS 109 THR 156 ILE 211 LEU 277 ARG37 LYS 110 LYS 158 GLU 212 ASP 278 SER 40 TYR 111 GLU 159 SER 213 PRO280 GLU 44 ASP 112 TYR 160 PRO 214 ASN 281 ARG 45 ASP 113 MET 161 THR215 LYS 282 TYR 46 LYS 114 SER 162 ASP 216 ALA 283 LYS 47 ASN 115 THR163 LYS 218 SER 285 ASP 48 HIS 116 LEU 164 LYS 221 SER 286 TRP 49 ASN117 THR 168 LYS 222 SER 287 GLU 50 LYS 118 TYR 170 ILE 224 PHE 288 LYS51 LYS 120 PHE 171 PHE 225 SER 289 GLU 56 ARG 121 ASN 172 ASN 226 PRO291 MET 62 ALA 122 GLY 173 ASN 227 ASP 292 THR 64 GLN 123 ASN 174 MET228 PHE 293 ASN *Position corresponds to the specific amino acidposition in SEQ ID NO: 14.

While the α-hemolysin variant can include various combinations ofsubstitutions as described herein, in certain example embodiments theα-hemolysin variant includes particular combinations of substitutions.For example, an α-hemolysin variant may include the followingcombinations of amino acid substitutions of the sequence set forth asSEQ ID NO: 14:

-   -   H35G+V149K;    -   V149K+E287R+H35G;    -   V149K+E287R;    -   T109K+H35G;    -   P151K+H35G;    -   V149K+P151K+H35G;    -   T109K+V149K+H35G;    -   V149K+K147N+E111N+127−131G+M113A+H35G;    -   V149K+K147N+E111N+127−131G+M113A; or,    -   T109K+V149K+P151K+H35G.        Such combinations may also include, for example, a substitution        at H144A of SEQ ID NO: 14 and/or a series of glycine residues at        amino acids 127-131 of SEQ ID NO: 14. In certain example        embodiments, the α-hemolysin variant includes an amino acid        sequence having at least 80%, 90%, 95%, 98%, or more sequence        identity to the amino acid sequence set forth as SEQ ID NO: 4,        SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID        NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID        NO: 13, with the substitution(s) identified in each sequence,        for example, being preserved in the variant.

So that the variants and WT alpha-hemolysin can be manipulated, incertain example embodiments any of the amino acid sequences describedherein, such as those set forth as SEQ ID NO: 4-14, may also include alinker/TEV/HisTAG sequence at the C-terminal end having the sequenceGLSAENLYFQGHHHHHH (SEQ ID NO: 16, where the TEV sequence is underlined).As those skilled in the art will appreciate, such a sequence allows forthe purification of the variant.

Nanopore Assembly and Insertion

The methods described herein can use a nanopore having a polymeraseattached to the nanopore. In certain example embodiments, it isdesirable to have one and only one polymerase per nanopore (e.g., sothat only one nucleic acid molecule is sequenced at each nanopore).However, many nanopores, including alpha-hemolysin (aHL), can bemultimeric proteins having a plurality of subunits (e.g., 7 subunits foraHL). The subunits can be identical copies of the same polypeptide.Provided herein are multimeric proteins (e.g., nanopores) having adefined ratio of modified subunits (e.g., a-HL variants) to un-modifiedsubunits (e.g., a-HL).

Also provided herein are methods for producing multimeric proteins(e.g., nanopores or nanopore assemblies) having a defined ratio ofmodified subunits to un-modified subunits. For example, the nanoporeassembly may include any of the alpha-hemolysin variants describedherein. A heptameric nanopore assembly, for example, may include one ormore alpha-hemolysin subunits having an amino acidic sequencecorresponding to a substitution of any one of V149K, E287R, H35G, T109K,P151K, K147N, E111N, M113A, or combinations thereof of SEQ ID NO: 14. Incertain example embodiments, one or more of the subunits may include aspecific combination of substitutions as described herein. Any of thevariants used in the nanopore assembly, such as in a heptamericassembly, may also include an H144A substitution of SEQ ID NO: 14.

With reference to FIG. 27 of WO2014/074727, a method for assembling aprotein having a plurality of subunits includes providing a plurality offirst subunits 2705 and providing a plurality of second subunits 2710,where the second subunits are modified when compared with the firstsubunits. In some cases, the first subunits are wild-type (e.g.,purified from native sources or produced recombinantly). The secondsubunits can be modified in any suitable way. In some cases, the secondsubunits have a protein (e.g., a polymerase) attached (e.g., as a fusionprotein).

In certain example embodiments, the modified subunits can comprise achemically reactive moiety (e.g., an azide or an alkyne group suitablefor forming a linkage). In some cases, the method further comprisesperforming a reaction (e.g., a Click chemistry cycloaddition) to attachan entity (e.g., a polymerase) to the chemically reactive moiety.

In certain example embodiments, the method can further includecontacting the first subunits with the second subunits 2715 in a firstratio to form a plurality of proteins 2720 having the first subunits andthe second subunits. For example, one part modified aHL subunits havinga reactive group suitable for attaching a polymerase can be mixed withsix parts wild-type aHL subunits (i.e., with the first ratio being 1:6).The plurality of proteins can have a plurality of ratios of the firstsubunits to the second subunits. For example, the mixed subunits canform several nanopores having a distribution of stoichiometries ofmodified to un-modified subunits (e.g., 1:6, 2:5, 3:4).

In certain example embodiments, the proteins are formed by simply mixingthe subunits. In the case of aHL nanopores for example, a detergent(e.g., deoxycholic acid) can trigger the aHL monomer to adopt the poreconformation. The nanopores can also be formed, for example, using alipid (e.g., 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or1,2-di-0-phytanyl-sn-glycero-3-phosphocholine (DoPhPC)) and moderatetemperature (e.g., less than about 100° C.). In some cases, mixing DPhPCwith a buffer solution creates large multi-lamellar vesicles (LMV), andadding aHL subunits to this solution and incubating the mixture at 40°C. for 30 minutes results in pore formation.

If two different types of subunits are used (e.g., the natural wild typeprotein and a second aHL monomer which can contain a single pointmutation), the resulting proteins can have a mixed stoichiometry (e.g.,of the wild type and mutant proteins). The stoichiometry of theseproteins can, in certain example embodiments, follow a formula which isdependent upon the ratio of the concentrations of the two proteins usedin the pore forming reaction. This formula is as follows:100P _(m)=100[n!/m!(n−m)!]·f _(mut) ^(m) ·f _(wt) ^(n˜m), where

-   -   P_(m)=probability of a pore having m number of mutant subunits    -   n=total number of subunits (e.g., 7 for aHL)    -   m=number of “mutant” subunits    -   f_(mut)=fraction or ratio of mutant subunits mixed together    -   f_(wt)=fraction or ratio of wild-type subunits mixed together

The method can further comprise fractionating the plurality of proteinsto enrich proteins that have a second ratio of the first subunits to thesecond subunits 2725. For example, nanopore proteins can be isolatedthat have one and only one modified subunit (e.g., a second ratio of1:6). However, any second ratio is suitable. A distribution of secondratios can also be fractionated such as enriching proteins that haveeither one or two modified subunits. The total number of subunitsforming the protein is not always 7 (e.g., a different nanopore can beused or an alpha-hemolysin nanopore can form having six subunits) asdepicted in FIG. 27 of WO2014/074727. In some cases, proteins havingonly one modified subunit are enriched. In such cases, the second ratiois 1 second subunit per (n−1) first subunits where n is the number ofsubunits comprising the protein.

The first ratio can be the same as the second ratio, however this is notrequired. In some cases, proteins having mutated monomers can form lessefficiently than those not having mutated subunits. If this is the case,the first ratio can be greater than the second ratio (e.g., if a secondratio of 1 mutated to 6 non-mutated subunits are desired in a nanopore,forming a suitable number of 1:6 proteins may require mixing thesubunits at a ratio greater than 1:6).

Proteins having different second ratios of subunits can behavedifferently (e.g., have different retention times) in a separation. Incertain example embodiments, the proteins are fractionated usingchromatography, such as ion exchange chromatography or affinitychromatography. Since the first and second subunits can be identicalapart from the modification, the number of modifications on the proteincan serve as a basis for separation. In some cases, either the first orsecond subunits have a purification tag (e.g., in addition to themodification) to allow or improve the efficiency of the fractionation.In some cases, a poly-histidine tag (His-tag), a streptavidin tag(Strep-tag), or other peptide tag is used. In some instances, the firstand second subunits each comprise different tags and the fractionationstep fractionates on the basis of each tag. In the case of a His-tag, acharge is created on the tag at low pH (Histidine residues becomepositively charged below the pKa of the side chain). With a significantdifference in charge on one of the aHL molecules compared to the others,ion exchange chromatography can be used to separate the oligomers whichhave 0, 1, 2, 3, 4, 5, 6, or 7 of the “charge-tagged” aHL subunits. Inprinciple, this charge tag can be a string of any amino acids whichcarry a uniform charge. FIG. 28 and FIG. 29 show examples offractionation of nanopores based on a His-tag. FIG. 28 shows a plot ofultraviolet absorbance at 280 nanometers, ultraviolet absorbance at 260nanometers, and conductivity. The peaks correspond to nanopores withvarious ratios of modified and unmodified subunits. FIG. 29 ofWO2014/074727 shows fractionation of aHL nanopores and mutants thereofusing both His-tag and Strep-tags.

In certain example embodiments, an entity (e.g., a polymerase) isattached to the protein following fractionation. The protein can be ananopore and the entity can be a polymerase. In some instances, themethod further comprises inserting the proteins having the second ratiosubunits into a bilayer.

In certain example embodiments, a nanopore can comprise a plurality ofsubunits. As described herein, a polymerase can be attached to one ofthe subunits and at least one and less than all of the subunits comprisea first purification tag. In some example embodiments, the nanopore isalpha-hemolysin or a variant thereof. In some instances, all of thesubunits comprise a first purification tag or a second purification tag.The first purification tag can, for example, be a poly-histidine tag(e.g., on the subunit having the polymerase attached).

Polymerase Attached to Nanopore

In certain example embodiments, a polymerase (e.g., DNA polymerase) isattached to and/or is located in proximity to the nanopore. Any DNApolymerase capable of synthesizing DNA during a DNA synthesis reactionmay be used in accordance with the methods and compositions describedherein. Example DNA polymerases include, but are not limited to, phi29(Bacillus bacteriophage φ29), pol6 (Clostridium phage phiCPV4; GenBank:AFH27113.1) or pol7 (Actinomyces phage Av-1; GenBank: ABR67671.1). Incertain example embodiments, attached to the nanopore assembly is aDNA-manipulating or modifying enzyme, such as a ligase, nuclease,phosphatase, kinase, transferase, or topoisomerase.

In certain example embodiments, the polymerase is a polymerase variant.For example, the polymerase variant may include any of the polymerasevariants identified in U.S. patent application Ser. No. 15/012,317 (the“317 Application”). Such variants include, for example, one or moreamino acid substitutions at H223A, N224Y/L, Y225L/T/I/F/A, H227P,I295W/F/M/E, Y342L/F, T343N/F, I357G/L/Q/H/W/M/A/E/Y/P, S360G,L361M/W/V, I363V, S365Q/W/M/A/G, S366A/L, Y367L/E/M/P/N, P368G, D417P,E475D, Y476V, F478L, K518Q, H527W/R/L, T529M/F, M531H/Y/A/K/R/W/T/L/V,N535L/Y/M/K/I, G539Y/F, P542E/S, N545K/D/S/L/R, Q546W/F,A547M/Y/W/F/V/S, L549Q/Y/H/G/R, 1550A/W/T/G/F/S, N552L/M/S, G553S/T,F558P/T, A596S, G603T, A610T/E, V615A/T, Y622A/M, C623G/S/Y, D624F,I628Y/V/F, Y629W/H/M, R632L/C, N635D, M641L/Y, A643L, I644H/M/Y,T647G/A/E/K/S, I648K/R/V/N/T, T651Y/F/M, I652Q/G/S/N/F/T, K655G/F/E/N,W656E, D657R/P/A, V658L, H660A/Y, F662I/L, L690M, or combinationsthereof of SEQ ID NO: 15 (which corresponds to SEQ ID NO: 2 of the '317Application). In certain example embodiments, the polymerase includesone or more such substitutions and has 80%, 90%, 95%, 98% or moresequence identity to the amino acid sequence set forth as SEQ ID NO: 15.As described in the '317 Application, the polymerase variant has alteredenzyme activity, fidelity, processivity, elongation rate, sequencingaccuracy, long continuous read capability, stability, or solubilityrelative to the parental polymerase.

The polymerase can be attached to the nanopore in any suitable way. Apolymerase, for example, can be attached to the nanopore assembly in anysuitable way known in the art. See, for example, PCT/US2013/068967(published as WO2014/074727; Genia Technologies), PCT/US2005/009702(published as WO2006/028508), and PCT/US2011/065640 (published asWO2012/083249; Columbia Univ). In certain example embodiments, thepolymerase is attached to the nanopore (e.g., hemolysin) protein monomerand then the full nanopore heptamer is assembled (e.g., in a ratio ofone monomer with an attached polymerase to 6 nanopore (e.g., hemolysin)monomers without an attached polymerase). The nanopore heptamer can thenbe inserted into the membrane.

Another method for attaching a polymerase to a nanopore involvesattaching a linker molecule to a hemolysin monomer or mutating ahemolysin monomer to have an attachment site and then assembling thefull nanopore heptamer (e.g., at a ratio of one monomer with linkerand/or attachment site to 6 hemolysin monomers with no linker and/orattachment site). A polymerase can then be attached to the attachmentsite or attachment linker (e.g., in bulk, before inserting into themembrane). The polymerase can also be attached to the attachment site orattachment linker after the (e.g., heptamer) nanopore is formed in themembrane. In some cases, a plurality of nanopore-polymerase pairs areinserted into a plurality of membranes (e.g., disposed over the wellsand/or electrodes) of the biochip. In some instances, the attachment ofthe polymerase to the nanopore complex occurs on the biochip above eachelectrode.

The polymerase can be attached to the nanopore with any suitablechemistry (e.g., covalent bond and/or linker). In some cases, thepolymerase is attached to the nanopore with molecular staples. In someinstances, molecular staples comprise three amino acid sequences(denoted linkers A, B and C). Linker A can extend from a hemolysinmonomer, Linker B can extend from the polymerase, and Linker C then canbind Linkers A and B (e.g., by wrapping around both Linkers A and B) andthus the polymerase to the nanopore. Linker C can also be constructed tobe part of Linker A or Linker B, thus reducing the number of linkermolecules.

In certain example embodiments, the polymerase is linked to the nanoporeusing Solulink™ chemistry. Solulink™ can be a reaction between HyNic(6-hydrazino-nicotinic acid, an aromatic hydrazine) and 4FB(4-formylbenzoate, an aromatic aldehyde). In some instances, thepolymerase is linked to the nanopore using Click chemistry (availablefrom LifeTechnologies for example). In some cases, zinc finger mutationsare introduced into the hemolysin molecule and then a molecule is used(e.g., a DNA intermediate molecule) to link the polymerase to the zincfinger sites on the hemolysin.

Additionally or alternatively, the SpyTag/SpyCatcher system, whichspontaneously forms covalent isopeptide linkages under physiologicalconditions, may be used to join an alpha-hemolysin monomer to thepolymerase. See, for example, Li et al, J Mol Biol. 2014 Jan. 23;426(2):309-17. For example, an alpha-hemolysin protein can be expressedhaving a SpyTag domain. Further, the DNA polymerase to be joined to thealpha-hemolysin may be separately expressed as fusion protein having aSpyCatcher domain. By mixing the alpha-hemolysin/SpyTag fusion proteinwith the DNA Polymerase/SpyCatcher protein, the SpyTag and SpyCatcherproteins interact to form the alpha-hemolysin monomer that is linked toa DNA polymerase via a covalent isopeptide linkage.

In certain example embodiments, the polymerase may be attached to ananopore monomer before the nanopore monomer is incorporated into ananopore assembly. For example, following expression and purification ofthe alpha-hemolysin/SpyTag fusion protein, the purifiedalpha-hemolysin/SpyTag fusion protein is mixed with purifiedpolymerase/SpyCatcher fusion protein, thus allowing the SpyTag andSpyCatcher proteins bind each other to form analpha-hemolysin/polymerase monomer. The monomer can then be incorporatedinto the nanopore assembly as described herein to form a heptamericassembly.

In certain example embodiments, the polymerase is attached to thenanopore assembly after formation of the nanopore assembly. For example,following expression and purification of the alpha-hemolysin/SpyTagfusion protein, the fusion protein is incorporated into the nanoporeassembly to form the heptameric nanopore assembly. Thepolymerase/SpyCatcher fusion protein is then mixed with the heptamericassembly, thus allowing the SpyTag and SpyCatcher proteins bind eachother, which in turn results in binding of the polymerase to thenanopore assembly.

Because of the nature of nanopore-based sequencing reaction, thoseskilled in the art will appreciate that it is beneficial to have only asingle polymerase associated with each nanopore assembly (rather thanmultiple polymerases). To achieve such assemblies, the nanopore assemblymay be configured, for example, to have only a single SpyTag, whichtherefore allows the attachment of a single polymerase/SpyCatcher.

In the case of alpha-hemolysin, for example, mixing thealpha-hemolysin/SpyTag proteins with additional alpha-hemolysin proteinsresults in heptamers having 0, 1, 2, 3, 4, 5, 6, or 7alpha-hemolysin/SpyTag subunits. Yet because of the different number ofSpyTag sequences (0, 1, 2, 3, 4, 5, 6, or 7) associated with eachheptamer, the heptamers have different charges. Hence, in certainexample embodiments, the heptamers can be separated by methods known inthe art, such as via elution with cation exchange chromatography. Theeluted fractions can then be examined to determine which fractionincludes an assembly with a single SpyTag. The fraction with a singleSpyTag can then be used to attach a single polymerase to each assembly,thereby creating a nanopore assemblies with a single polymerase attachedthereto.

While a variety of methods may be suitable for determining whichheptamer fraction contains a single SpyTag (and that is hence capable ofbinding a only single polymerase/SpyCatcher fusion protein perheptamer), in certain example embodiments the different heptamerfraction can be separated based on molecular weight, such as viaSDS-PAGE. A reagent can then be used to confirm the presence of SpyTagassociated with each fraction. For example, a SpyCatcher-GFP (greenfluorescent protein) can be added to the fractions before separation viaSDS-PAGE.

Because heptamers with fewer number of SpyTags are smaller than theheptamers with greater number of SpyTags, the fraction with a singleSpyTag can be identified, as evidenced by the furthest band migrationand the presence of GFP fluorescence in the SDS-PAGE gel correspondingto the band. For example, a fraction containing seven alpha-hemolysinmonomers and zero SpyTag fusion proteins will migrate the furthest, butwill not fluoresce when mixed with SpyCatcher-GFP because of the absenceof the SpyTag bound to the heptamers. The fraction containing a singleSpyTag, however, will both migrate the next furthest (compared to otherfluorescent bands) and will fluoresce, thereby allowing identificationof the fraction with a single SpyTag bound to the heptamer. Followingidentification of the fraction with a single SpyTag bound to theheptamer, the polymerase/SpyCatcher fusion protein can then be added tothis fraction, thereby linking the polymerase to the nanopore assembly.

By using the methods and compositions described herein, a nanoporeassembly tethered to a single DNA polymerase—and including one or moreof the alpha hemolysin variants as described herein—can be achieved. Forexample, the heptameric nanopore may include one alpha-hemolysin varianthaving a substitution corresponding to any one of V149K, E287R, H35G,T109K, P151K, K147N, E111N, M113A, or combinations thereof of SEQ ID NO:14, five mature wild type alpha hemolysin monomers, and a seventhalpha-hemolysin monomer that is fused to a polymerase (for a total ofseven subunits of the heptamer). In certain example embodiments, thenanopore heptamer assembly may include 1, 2, 3, 4, 5, 6, or 7 of thevariants described herein, with one of the subunits being attached to apolymerase as described herein.

Apparatus Set-Up

The nanopore may be formed or otherwise embedded in a membrane disposedadjacent to a sensing electrode of a sensing circuit, such as anintegrated circuit. The integrated circuit may be an applicationspecific integrated circuit (ASIC). In some examples, the integratedcircuit is a field effect transistor or a complementary metal-oxidesemiconductor (CMOS). The sensing circuit may be situated in a chip orother device having the nanopore, or off of the chip or device, such asin an off-chip configuration. The semiconductor can be anysemiconductor, including, without limitation, Group IV (e.g., silicon)and Group III-V semiconductors (e.g., gallium arsenide). See, forexample, WO 2013/123450, for the apparatus and device set-up for sensinga nucleotide or tag.

Pore based sensors (e.g., biochips) can be used forelectro-interrogation of single molecules. A pore based sensor caninclude a nanopore of the present disclosure formed in a membrane thatis disposed adjacent or in proximity to a sensing electrode. The sensorcan include a counter electrode. The membrane includes a trans side(i.e., side facing the sensing electrode) and a cis side (i.e., sidefacing the counter electrode).

In certain example embodiments, a nanopore including one or more of thealpha-hemolysin variants described herein, will have an altered time tothread relative to a nanopore including wild-type alpha-hemolysin (i.e.,a nanopore without any of the substitutions described herein). Forexample, the time for a tag to thread through the pore (thetime-to-thread or TTT) may be decreased. In certain example embodiments,the TTT for a nanopore comprising one or more of the variants describedherein may be decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%or more as compared to a heptameric nanopore assembly consisting ofnative alpha-hemolysin.

In the experimental disclosure that follows, the following abbreviationsapply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol(moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g(grams); mg (milligrams); kg (kilograms); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); h (hours);min (minutes); sec (seconds); msec (milliseconds).

EXAMPLES

The present invention is described in further detain in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein. The following examples are offered toillustrate, but not to limit the claimed invention.

Example 1 Expression and Recovery

This example illustrates the expression and recovery of protein frombacterial host cells, e.g., E. coli.

DNA encoding the wild-type a-HL was purchased from a commercial source.The sequence was verified by sequencing.

Plasmid Construction.

The gene encoding either a wild-type or variant α-nemolysin was insertedinto a pPR-IBA2 plasmid (IBA Life Sciences, Germany) under the controlof T7 promoter.

Transformation.

E. coli BL21 DE3 (from Life Technologies) cells were transformed withthe expression vector comprising the DNA encoding the wild-type orvariant α-hemolysin using techniques well-known in the art. Briefly, thecells were thawed on ice (if frozen). Next, the desired DNA (in asuitable vector/plasmid) was added directly into the competent cells(should not exceed 5% of that of the competent cells) and mixed byflicking the tube. The tubes were placed on ice for 20 minutes. Next,the cells were placed in a 42° C. water bath for 45 seconds withoutmixing, followed by placing the tubes on ice for 2 min. The cells werethen transferred to a 15 ml sterilized culture tube containing 0.9 ml ofSOC medium (pre-warmed at room temperature) and cultured at 37° C. for 1hr in a shaker. Finally, an aliquot of the cells were spread onto a LBagar plate containing the appropriate antibiotic and the platesincubated at 37° C. overnight.

Protein Expression.

Following transformation, colonies were picked and inoculated into asmall volume (e.g., 3 ml) of growth medium (e.g., LB broth) containingthe appropriate antibiotic with shaking at 37° C., overnight.

The next morning, transfer 1 ml of the overnight culture to a new 100 mlof autoinduction medium, e.g., Magic Media (Life Technologies)containing an appropriate antibiotic to select the expression plasmid.Grow the culture with shaking at 25° C. approximately 16 hrs but thisdepended on the expression plasmids. Cells were harvested bycentrifugation at 3,000 g for 20 min at 4° C. and stored at −80° C.until used.

Purification.

Cells were lysed via sonication. The alpha-hemolysin was purified tohomogeneity by affinity column chromatography.

Example 2 Alpha-Hemolysin Variants

The following example details the introduction of a mutation at adesired residue.

Mutations.

Site-directed mutagenesis is carried out using a QuikChange MultiSite-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) to preparethe example H35G+V149K+H144A, as set forth in SEQ ID NO: 4, but alsoincluding a C-terminal linker/TEV/HisTag for purification.

The variant was expressed and purified as in Example 1.

Example 3 Assembly of Nanopore Including Variant

This example describes the assembly of a nanopore comprising six a-HLvariant subunits and one wild-type subunit.

The wild-type a-HL was expressed as described in Example 1 with SpyTagand a HisTag and purified on a cobalt affinity column using a cobaltelution buffer (200 mM NaCl, 300 mM imidazole, 50 mM tris, pH 8). TheH35G+V149K+H144A a-HL variant was expressed as described in Example 1with a HisTag and purified on a cobalt affinity column using a cobaltelution buffer (200 mM NaCl, 150 mM imidazole, 50 mM tris, pH 8). Theprotein was then incubated with 1 mg of TEV protease for every 5 mg ofprotein at 4 C for 4 hours. After incubation with TEV protease themixture is purified on a cobalt affinity column to remove TEV proteaseand undigested protein. The proteins were stored at 4° C. if used within5 days, otherwise 8% trehalose was added and stored at −80° C.

Using approximately 10 mg of total protein, the α-HL/SpyTag to desiredα-HL-variant protein solutions were mixed together at a 1:9 ratio toform a mixture of heptamers. It is expected that such a mixture willresult in various fractions that include varying ratios of α-HL/SpyTagand α-HL-variant protein (0:7; 1:6, 2:5, 3:4, etc.), where the SpyTagcomponent is present as 0, 1, 2, 3, 4, 5, 6, or seven monomeric subunitsof the heptamer.

Diphytanoylphosphatidylcholine (DPhPC) lipid was solubilized in either50 mM Tris, 200 mM NaCl, pH 8 or 150 mM KCl, 30 mM HEPES, pH 7.5 to afinal concentration of 50 mg/ml and added to the mixture of a-HLmonomers to a final concentration of 5 mg/ml. The mixture of the α-HLmonomers was incubated at 37° C. for at least 60 min. Thereafter,n-Octyl-β-D-Glucopyranoside (βOG) was added to a final concentration of5% (weight/volume) to solubilize the resulting lipid-protein mixture.The sample was centrifuged to clear protein aggregates and left overlipid complexes and the supernatant was collected for furtherpurification.

The mixture of heptamers was then subjected to cation exchangepurification and the elution fractions collected. For each fraction, twosamples were prepared for SDS-PAGE. The first sample included 15 uL ofα-HL eluate alone and the second sample was combined with 3 ug ofSpyCatcher-GFP. The samples were then incubated and sheltered from lightand at room temperature for 1-16 hours. Following incubation, 5 uL of 4×Laemmli SDS-PAGE buffer (Bio-Rad™) was added to each sample. The samplesand a PrecisionPlus™ Stain-Free protein ladder were then loaded onto a4-20% Mini-PROTEAN Stain-Free protein precast gel (Bio-Rad). The gelswere ran at 200 mV for 30 minutes. The gels were then imaged using aStain-Free filter.

The conjugation of SpyCatcher-GFP to heptameric α-HL/SpyTag can beobserved through molecular weight band shifts during SDS-PAGE. Heptamerscontaining a single SpyTag will bind a single SpyCatcher-GFP molecularand will thus have a shift that corresponds to the molecular weight ofthe heptameric pore plus the molecular weight of a singleSpyCatcher-GFP, while heptamers with two or more SpyTags should havecorrespondingly larger molecular weight shifts. Therefore, the peakseluted off of the cation exchange column during heptameric α-HLpurification above can be analyzed for the ratio of α-HL/SpyTag toα-HL-variant. In addition, the presence of SpyCatcher-GFP attachment canbe observed using a GFP-fluorescence filter when imaging the SDS-PAGEgels.

Based on this rationale, the fraction whose molecular weight shiftcorresponded to a single addition of SpyCatcher-GFP was determined usinga molecular weight standard protein ladder. Bio-Rad's stain-free imagingsystem was used to determine the molecular weight shift. The presence ofGFP fluorescence was determined using a blue filter. The presence offluorescence was used to confirm the presence of the SpyTag protein. Theelution fraction corresponding to the 1:6 ratio, i.e., one α-HL/SpyTagto six α-HL-variants, was then used for further experiments.

Example 4 Attachment of a Polymerase

This example provides for the attachment of a polymerase to a nanopore.

The polymerase may be coupled to the nanopore by any suitable means.See, for example, PCT/US2013/068967 (published as WO2014/074727; GeniaTechnologies), PCT/US2005/009702 (published as WO2006/028508), andPCT/US2011/065640 (published as WO2012/083249; Columbia Univ).

The polymerase, e.g., phi29 DNA Polymerase, was coupled to a proteinnanopore (e.g. alpha-hemolysin), through a linker molecule.Specifically, the SpyTag and SpyCatcher system, which spontaneouslyforms covalent isopeptide linkages under physiological conditions, wasused. See, for example, Li et al, J Mol Biol. 2014 Jan. 23;426(2):309-17.

Briefly, the Sticky phi29 SpyCatcher HisTag was expressed according toExample 1 and purified using a cobalt affinity column. The SpyCatcherpolymerase and the SpyTag oligomerized protein were incubated at a 1:1molar ratio overnight at 4° C. in 3 mM SrCl₂. The1:6-polymerase-template complex is then purified using size-exclusionchromatography.

Example 5 Activity of the Variant

This example shows the activity of the nanopores as provided by Example4 (nanopores with an attached polymerase).

The wild-type and H35G+V149K+H144A variant nanopores were assayed todetermine the effect of the substitutions. More particularly, the assaywas designed to measure the time it takes to capture a tagged moleculeby a DNA polymerase attached to the nanopore using alternating voltages,i.e., squarewaves.

The bilayers were formed and pores were inserted as described inPCT/US14/61853 filed Oct. 23, 2014. The nanopore device (or sensor) usedto detect a molecule (and/or sequence a nucleic acid) was set-up asdescribed in WO2013123450.

To measure the time it takes to capture a tagged nucleotide by a DNApolymerase in our sequencing complex we have devised an assay that usesalternating positive and negative voltages (squarewaves) to determinethe amount of time this takes. Our sequencing complex is comprised of aprotein nanopore (aHL) which is attached to a single DNA polymerase (seeExample 4). The tagged nucleotides are negatively charged, and aretherefore attracted to the nanopore when the voltage applied is positivein nature, and repelled when the voltage applied to the nanoporesequencing complex is negative. So we can measure the time it takes fora tag to thread into the pore by cycling the voltage between positiveand negative potentials and determine how much time the nanopore'scurrent is unobstructed (open channel) verses when the tag is threaded(reduced current flux).

To carry out the “time-to-thread” assay, the Genia Sequencing device isused with a Genia Sequencing Chip. The electrodes are conditioned andphospholipid bilayers are established on the chip as explained inPCT/US2013/026514. Genia's sequencing complex is inserted to thebilayers following the protocol described in PCT/US2013/026514(published as WO2013/123450). The time-to-thread data was collectedusing a buffer system comprised of 20 mM HEPES pH 8, 300 mM KGlu, 3 uMtagged nucleotide, 3 mM Mg²⁺, with a voltage applied of 235 mV peak topeak with a duty cycle of 80 Hz.

After the data was collected, it was analyzed for squarewaves thatshowed the capture of a tagged nucleotide (threaded level) which lastedto the end of the positive portion of the squarewave, and was followedby another tag capture on the subsequent squarewave. The time-to-threadwas measured by determining how long the second squarewave reportedunobstructed open channel current. As an example, if 10 consecutivesquarewaves showed tagged nucleotide captures that lasted to the end ofthe positive portion of the squarewave then the time-to-thread parameterwould be calculated from squarewaves 2-10 (the first squarewave does notfactor into the calculation because the polymerase did not have a tagbound to it in the previous squarewave). These time-to-thread numberswere then collected for all of the pores in the experiment andstatistical parameters extracted from them (such as a mean, median,standard deviation etc.).

Results for the H35G+V149K+H144A variant, as compared to controls, areshown in FIG. 1-2.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

SEQUENCE LISTING FREE TEXT SEQ ID NO: 1 (WT aHL DNA)ATGGCAGATC TCGATCCCGC GAAATTAATA CGACTCACTA TAGGGAGGCC   50ACAACGGTTT CCCTCTAGAA ATAATTTTGT TTAACTTTAA GAAGGAGATA  100TACAAATGGA TTCAGATATT AATATTAAAA CAGGTACAAC AGATATTGGT  150TCAAATACAA CAGTAAAAAC TGGTGATTTA GTAACTTATG ATAAAGAAAA  200TGGTATGCAT AAAAAAGTAT TTTATTCTTT TATTGATGAT AAAAATCATA  250ATAAAAAATT GTTAGTTATT CGTACAAAAG GTACTATTGC AGGTCAATAT  300AGAGTATATA GTGAAGAAGG TGCTAATAAA AGTGGTTTAG CATGGCCATC  350TGCTTTTAAA GTTCAATTAC AATTACCTGA TAATGAAGTA GCACAAATTT  400CAGATTATTA TCCACGTAAT AGTATTGATA CAAAAGAATA TATGTCAACA  450TTAACTTATG GTTTTAATGG TAATGTAACA GGTGATGATA CTGGTAAAAT  500TGGTGGTTTA ATTGGTGCTA ATGTTTCAAT TGGTCATACA TTAAAATATG  550TACAACCAGA TTTTAAAACA ATTTTAGAAA GTCCTACTGA TAAAAAAGTT  600GGTTGGAAAG TAATTTTTAA TAATATGGTT AATCAAAATT GGGGTCCTTA  650TGATCGTGAT AGTTGGAATC CTGTATATGG TAATCAATTA TTTATGAAAA  700CAAGAAATGG TTCTATGAAA GCAGCTGATA ATTTCTTAGA TCCAAATAAA  750GCATCAAGTT TATTATCTTC AGGTTTTTCT CCTGATTTTG CAACAGTTAT  800TACTATGGAT AGAAAAGCAT CAAAACAACA AACAAATATT GATGTTATTT  850ATGAACGTGT AAGAGATGAT TATCAATTAC ATTGGACATC AACTAATTGG  900AAAGGTACAA ATACTAAAGA TAAATGGACA GATAGAAGTT CAGAAAGATA  950TAAAATTGAT TGGGAAAAAG AAGAAATGAC AAATGGTCTC AGCGCTTGGA 1000GCCACCCGCA GTTCGAAAAA TAA 1023SEQ ID NO: 2 (WT aHL amino acids) [as expressed in E. coli]MADSDINIKT GTTDIGSNTT VKTGDLVTYD KENGMHKKVF YSFIDDKNHN  50KKLLVIRTKG TIAGQYRVYS EEGANKSGLA WPSAFKVQLQ LPDNEVAQIS 100DYYPRNSIDT KEYMSTLTYG FNGNVTGDDT GKIGGLIGAN VSIGHTLKYV 150QPDFKTILES PTDKKVGWKV IFNNMVNQNW GPYDRDSWNP VYGNQLFMKT 200RNGSMKAADN FLDPNKASSL LSSGFSPDFA TVITMDRKAS KQQTNIDVIY 250ERVRDDYQLH WTSTNWKGTN TKDKWTDRSS ERYKIDWEKE EMTNGLSAWS 300 HPQFEK 306SEQ ID NO: 3 (Mature WT aHL, with purification tag)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTNGLSAWSH 300 PQFEK 305SEQ ID NO: 4 (H35G + V149K + H144A)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIG A TLKY K Q 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293 SEQ ID NO: 5 (H35G +H144A + V149K + E287R) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM GKKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIG A TLKY K Q 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDW R KEE MTN 293SEQ ID NO: 6 (V149K + E287R)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMGKKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKY K Q 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDW R KEE MTN 293SEQ ID NO: 7 (T109 + H35G) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM GKKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSID KK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGATLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293SEQ ID NO: 8 (P151K + H35G) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM GKKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGATLKYVQ 150 KDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293SEQ ID NO: 9 (V149K + P151K + H35G)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGATLKY K Q 150 KDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293SEQ ID NO: 10 (T109K + V149K + H35G)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSID KK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGATLKY K Q 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293  SEQ ID NO: 11 (V149K + K147N + E111N + 127  131 + M113A  +  H35G)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK  NY A STLTYGF NGNVT GGGGG   G IGGLIGANV SIGATL N Y K Q 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293SEQ ID NO: 12 (V149K + K147N + E111N + 127 - 131G + M113A)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK  NY A STLTYGF NGNVT GGGGG   G IGGLIGANV SIGHTL N Y K Q 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293SEQ ID NO: 13 (T109K + V149K + P151K + H35G)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSID KK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGATLKY K Q 150 KDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293SEQ ID NO: 14 (Mature WT aHL; AAA26598)ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK  50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293SEQ ID NO: 15 (13016 with His Tag)MHHHHHHHHS GGSDKHTQYV KEHSFNYDEY KKANFDKIEC LIFDTESCTN  50YENDNTGARV YGWGLGVTRN HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT 100IKITKTKKGF PKRKYIKFPI AVHNLGWDVE FLKYSLVENG FNYDKGLLKT 150VFSKGAPYQT VTDVEEPKTF HIVQNNNIVY GCNVYMDKFF EVENKDGSTT 200EIGLCLDFFD SYKIITCAES QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH 250KQTTLELRYQ YNDIYMLREV IEQFYIDGLC GGELPLTGMR TASSIAFNVL 300KKMTFGEEKT EEGYINYFEL DKKTKFEFLR KRIEMESYTG GYTHANHKAV 350GKTINKIGCS LDINSSYPSQ MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI 400EVGFDFVEPK HEEYALDIFK IGAVNSKALS PITGAVSGQE YFCTNIKDGK 450AIPVYKELKD TKLTTNYNVV LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN 500LEFTGLKIGS ILYYKAEKGK FKPYVDHFTK MKVENKKLGN KPLTNQAKLI 550LNGAYGKFGT KQNKEEKDLI MDKNGLLTFT GSVTEYEGKE FYRPYASFVT 600AYGRLQLWNA IIYAVGVENF LYCDTDSIYC NREVNSLIED MNAIGETIDK 650TILGKWDVEH VFDKFKVLGQ KKYMYHDCKE DKTDLKCCGL PSDARKIIIG 700QGFDEFYLGK NVEGKKQRKK VIGGCLLLDT LFTIKKIMF* 739SEQ ID NO: 16 (Linker/TEV/HisTag (TEV portion underlined)GLSAENLYFQGHHHHHH

CITATION LIST Patent Literature

-   [1] PCT/US2013/026514 (published as WO2013/123450) entitled “Methods    for Creating Bilayers for Use with Nanopore Sensors”-   [2] PCT/US2013/068967 (published as WO 2014/074727) entitled    “Nucleic Acid Sequencing Using Tags”-   [3] PCT/US14/61853 filed Oct. 23, 2014 entitled “Methods for Forming    Lipid Bilayers on Biochips”

Non-Patent Literature

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The entirety of each patent, patent application, publication, document,GENBANK sequence, website and other published material referenced hereinhereby is incorporated by reference, including all tables, drawings, andfigures. All patents and publications are herein incorporated byreference to the same extent as if each was specifically andindividually indicated to be incorporated by reference. Citation of theabove patents, patent applications, publications and documents is not anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents. All patents and publications mentioned hereinare indicative of the skill levels of those of ordinary skill in the artto which the invention pertains.

What is claimed is:
 1. An α-hemolysin (α-HL) variant having at least 80%sequence identity to SEQ ID NO: 14 and comprising an amino acidsubstitution corresponding to any one of T109K, V149K, P151K, E287R, orcombinations thereof in SEQ ID NO:
 14. 2. The α-hemolysin variantaccording to claim 1, wherein the variant has a sequence having at least90%, 95%, 98%, or more sequence identity to the sequence set forth asSEQ ID NO:
 14. 3. The α-hemolysin variant according to claim 1, whereinthe variant further comprises a substitution at H144A of SEQ ID NO: 14.4. The α-hemolysin variant according to claim 1, wherein the variantfurther comprises two or more glycine residues at residues 127 through131.
 5. The α-hemolysin variant according to claim 1, wherein thesubstitution is V149K.
 6. The α-hemolysin variant according to claim 1,wherein the substitution is E287R.
 7. The α-hemolysin variant to claim1, wherein the substitution is T109K.
 8. The α-hemolysin variant toclaim 1, wherein the substitution is P151K.
 9. The α-hemolysin variantto claim 1, wherein the variant further comprises one or moresubstitutions substitution is selected from the group consisting ofH35G, E111N, M113A, H144A and K147N of SEQ ID NO:
 14. 10. Theα-hemolysin variant according to claim 9, wherein the variant comprisesamino acid substitutions H35G, H144A, and V149K (SEQ ID NO: 4).
 11. Theα-hemolysin variant according to claim 9, wherein the variant comprisesamino acid substitutions H35G, H144A, V149K, and E287R (SEQ ID NO: 5).12. The α-hemolysin variant according to claim 9, wherein the variantcomprises amino acid substitutions V149K and E287R (SEQ ID NO: 6). 13.The α-hemolysin variant according to claim 9, wherein the variantcomprises amino acid substitutions H35G and T109K (SEQ ID NO: 7). 14.The α-hemolysin variant according to claim 9, wherein the variantcomprises amino acid substitutions H35G and P151K (SEQ ID NO: 8). 15.The α-hemolysin variant according to claim 9, wherein the variantcomprises amino acid substitutions H35G, V149K, and P151K (SEQ ID NO:9).
 16. The α-hemolysin variant according to claim 9, wherein thevariant comprises amino acid substitutions H35G, T109K, and V149K (SEQID NO: 10).
 17. The α-hemolysin variant according to claim 9, whereinthe variant comprises amino acid substitutions H35G, E111N, M113A,K147N, and V149K and further comprising glycine at each of residues 127through 131 (SEQ ID NO: 11).
 18. The α-hemolysin variant according toclaim 9, wherein the variant comprises amino acid substitutions E111N,M113A, K147N, and V149K and further comprising glycine at each ofresidues 127 through 131 (SEQ ID NO: 12).
 19. The α-hemolysin variantaccording to claim 9, wherein the variant comprises amino acidsubstitutions H35G, T109K, V149K, and P151K (SEQ ID NO: 10).
 20. Theα-hemolysin variant according to any of claims 1, 3-6, 7-9, 2, and10-19, wherein the α-hemolysin variant is covalently bound to a DNApolymerase.
 21. The α-hemolysin variant according to claim 20, whereinsaid variant is bound to the DNA polymerase via an isopeptide bond. 22.A heptameric nanopore assembly comprising at least one α-hemolysinvariant according to any one of claims 1, 3-6, 7-9, 2, and 10-19. 23.The heptameric nanopore assembly according to claim 22, wherein the timeto thread (TTT) is decreased.
 24. The heptameric nanopore assemblyaccording to claim 23, wherein the TTT is decreased by about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% or more as compared to a heptamericnanopore assembly consisting of native alpha-hemolysin.
 25. A nucleicacid encoding the α-hemolysin variant according to any one of claims 1,3-6, 7-9, 2, and 10-19.
 26. The nucleic acid molecule of claim 25,wherein said nucleic acid molecule is derived from Staphylococcus aureus(SEQ ID NO: 1).
 27. A vector comprising a nucleic acid encoding analpha-hemolysin variant according to claim
 25. 28. A host celltransformed with the vector of claim
 27. 29. A method of producing anα-hemolysin variant comprising the steps of: (a) culturing the host cellaccording to claim 28 in a suitable culture medium under suitableconditions to produce alpha-hemolysin variant and (b) obtaining saidproduced alpha-hemolysin variant.
 30. A method for detecting a targetmolecule, comprising: (a) providing a chip comprising a heptamericnanopore assembly according to claim 22 in a membrane that is disposedadjacent or in proximity to a sensing electrode; (b) directing a nucleicacid molecule through said nanopore, wherein said nucleic acid moleculeis associated with a reporter molecule, wherein said nucleic acidmolecule comprises an address region and a probe region, wherein saidreporter molecule is associated with said nucleic acid molecule at saidprobe region, and wherein said reporter molecule is coupled to a targetmolecule; (c) sequencing said address region while said nucleic acidmolecule is directed through said nanopore to determine a nucleic acidsequence of said address region; and (d) identifying, with the aid of acomputer processor, said target molecule based upon a nucleic acidsequence of said address region determined in (c).